PROJECT SUMMARY/ABSTRACT: Telomere lengths and maintenance play very important roles in cancer, aging and other human diseases. The lengths of human telomeres are known to be highly heterogeneous and variable for different cell types/age and individuals, ranging from 0.5 kb to 20 kb in normal cells, and usually much shorter in most cancer cells. It is also known that the shortest telomere, not the average telomere length, is critical for cell viability and chromosome stability, and individual telomere length heterogeneity and telomere-driven genomic instability may contribute to early carcinogenesis. However, the most common methods (TRF, qPCR and Q-FISH) for telomere length measurements can only be used to estimate or infer the average length of the telomeres from many cells with very limited sensitivity, resolution and accuracy. Even more surprisingly, none is capable of measuring the length of the telomeres of all individual chromosomes and of single cells. The deficiency has greatly hammered many telomere-related association studies and the use of telomere lengths as biomarkers for clinical diagnosis of cancer and for prognosis of cancer treatments. To move the field of telomere biology forward and to enable the routine clinical use of telomeres as cancer biomarkers, we need a scalable technology for low-cost absolute length measurements of the telomeres of individual chromosomes in single cells. In this project, we propose to develop a technology (TeloMeSeq) for measuring the absolute lengths of the individual telomeres of all chromosomes in single human cells with single-nucleotide resolution in a scalable and economic manner. We will leverage on our NEM-SDA (nicking endonuclease mediated strand- displacement amplification) technology that has demonstrated to be capable of linear amplification of very genomic DNA molecules independent of length to develop a one-tube procedure for the unbiased linear amplification of all telomeres of single human cell and subsequent efficient sequencing library construction. We will also leverage the powerful Pacific Biosciences' single-molecule real-time sequencing technology (PacBio SMRT) that offers high throughput sequencing with very long reads (median read lengths > 20 kb) to sequence the amplified telomeres in full length to identify the individual telomeres and to determine their lengths by counting the number of telomere AATGGG repeats. We aim to demonstrate a proof of concept of the TeloMeSeq technology using single cells from normal human leukocytes and IMR90 cell line, and cancer cell lines (Jurkat and LNCaP). If successfully developed, our technology will enable the absolute length measurements of all individual telomeres in single human cells with single-base resolution, far superior to existing methods. Our TeloMeSeq technology has a great potential to transform telomere biology studies and clinical practice where telomeres are used as biomarkers for cancer diagnosis and staging, aging and other human diseases.